基于CRISPR/Cas9技术创建甜菜BvCENH3基因突变体的研究

Research on the construction of sugar beet BvCENH3 gene mutants based on CRISPR/Cas9 technology

【目的】通过CRISPR/Cas9技术对甜菜BvCENH3基因进行编辑,建立甜菜高效基因组编辑技术体系。【方法】以BvCENH3为编辑目标,设计双靶标构建基因编辑载体,通过农杆菌侵染甜菜叶柄获得转基因植株,利用二代测序技术检测转基因植株突变情况,通过微滴数字PCR技术筛选低拷贝编辑植株。【结果】获得82株甜菜转基因植株,其中40株被成功编辑,编辑效率为48.78%,靶标1效率优于靶标2。突变类型有单碱基替换(T→G、A→C)、碱基缺失(TC、TCTC缺失)等5种。筛选出23株低拷贝编辑植株,BvCENH3插入拷贝数为1.1~1.9。【结论】成功对甜菜BvCENH3进行定点编辑,获得40株BvCENH3基因突变体。初步创建了甜菜基因组编辑技术体系,为甜菜单倍育种奠定了理论与技术基础。

【Objective】The CRISPR/Cas9 technology was used to edit the BvCENH3 gene in sugar beet aiming to establish an efficient genome editing system.【Methods】Taking sugar beet BvCENH3 gene as the editing target,double candidate targets were selected to construct gene editing vector.Transgenic sugar beet plants were produced through agrobacterium-mediated transformation.Next generation sequencing technology was utilized to identify the mutation types and droplet digital PCR was employed to screen low-copy mutant plants.【Results】82 transgenic sugar beet plants were obtained,40 of which were successfully edited,with an editing efficiency of 48.78%.Target 1 was more efficient than target 2.There were five mutation types,including single base substitution(T→G、A→C)and base deletion(TC、TCTC deletion).23 low-copy edited plants were selected,with BvCENH3 insertion copy numbers ranging from 1.1 to 1.9.【Conclusion】Successfully edited BvCENH3 in sugar beet with 40 BvCENH3 gene mutants obtained.Preliminary established sugar beet genome editing system,laying theoretical and technical foundations for sugar beet haploid breeding.